In the past decade urea has surpassed and nearly replaced ammonium nitrate as a fertilizer. This has brought about new questions on urea and its use. Biuret [(H2N-CO-)2NH] is one of the impurities present in urea and can cause agronomic problems if placed near the seed or even if added as preplant in bands where seeds will later be planted. Therefore, accurate quantitative estimation of biuret in these fertilizers is extremely essential. The principle that lies behind a positive biuret test is same as that for a detecting a peptide bond. In the presence of peptides, a copper (II) ionforms a violet-colored complex in an alkaline solution. Similarly in presence of biuret, the copper (II) ion forms a violet colored chelate complex. In ICP, concentration of copper ion in this complex can be measured directly for the urea sample from which biuret concentration can be estimated based on the calibration curve drawn using the standard biuret solutions of different known concentrations.

Reagents prepared:

a)    Biuret standard dissolved in water (1000 mg/L)

b)    Urea sample dissolved in water (1 gm)

c)     7.5 g Copper sulphate pentahydrate dissolved in 500 ml water

d)    KOH buffer made by dissolving 12.3 g KOH and 15 g KCl in 500 ml water


Different volume of standard biuret solution (5, 10, 15, 20 and 25 ml) was pipetted out in different 250 ml beaker. Along with them, 1 gm of urea sample solution was also taken separately. As there was no available urea standard sample specifying certified value of biuret, synthetic standard samples were made by adding 10 ml and 20 ml of biuret standard solution (from 100 mg/L stock solution of biuret) in individual 1g of urea samples separately in other beakers. 25 ml of ethyl alcohol was added to each solution with stirring followed by addition of 10 ml copper sulphate and 20 ml of buffer solution to each during vigorous stirring. The excess copper precipitated as copper hydroxide was filtered out in 41 filter paper using pulp and in each case the volume was made upto 250 ml after thorough washing with water and ethanol. The concentration of copper in the purple colored copper-biuret complex was determined from ICP-AES at the wavelength of 327.393 nm. The calibration curve made using different standard biuret solution was plotted considering the biuret concentration in x axis and respective copper concentration in y axis. The same is shown in Fig.1 below. The copper concentrations were similarly measured in the unknown sample and from the calibration curve the biuret concentration was determined against the measured copper concentration. The biuret concentration determined for the unknown urea sample was found to be 0.20% (blue line). The repeatability of the obtained result was verified after determining the biuret concentration of the synthetic standard described earlier (red lines). Table 1 shows the comparison of the results along with the % recovery of biuret in the urea sample.


Table 1: Determined wt% concentration of biuret in urea and synthetic standard samples


Sample Details Measured Biuret conc in wt % Actual Biuret conc in wt % % Recovery of Biuret
Unknown Urea sample 0.20
Urea + 40 ppm Biuret 0.589 0.600 98.17
Urea + 80 ppm Biuret 0.982 1.00 98.20




Contributed by : Dr. (Mrs.) Saswati Ghosh